Cell growth substrates with tethered cell growth effector molecules

ABSTRACT

Disclosed are compositions with tethered growth effector molecules, and methods of using these compositions for growing cells and tissues. Growth effector molecules, including growth factors and extracellular matrix molecules, are flexibly tethered to a solid substrate. The compositions can be used either in vitro or in vivo to grow cells and tissues. By tethering the growth factors, they will not diffuse away from the desired location. By making the attachment flexible, the growth effector molecules can more naturally bind to cell surface receptors. A significant feature of these compositions and methods is that they enhance the biological response to the growth factors. The new method also offers other advantages over the traditional methods, in which growth factors are delivered in soluble form: (1) the growth factor is localized to a desired target cell population; (2) significantly less growth factor is needed to exert a biologic response. This method can be used for enhancing the therapeutic use of growth factors in vivo and creating surfaces which will enhance in vitro growth of difficult-to-grow cells such as liver cells.

The government has certain rights in the invention since this inventionwas made with government support under Grant Number 9157321-BCS awardedby the National Science Foundation.

This application is a continuation of U.S. Ser. No. 08/398,555 filedMar. 3, 1995.

BACKGROUND OF THE INVENTION

This invention concerns cell and tissue growth substrates, growthstimulation compositions, and methods for delivering growth factors tocells and tissues.

Long-term mammalian cell culture has been difficult to achieve. Manytypes of specialized cells plated on standard tissue culture plasticdishes dedifferentiate, lose function, and fail to proliferate. Thereare many applications of mammalian cell culture that could benefit frommethods or materials which enhance the long term stability ofdifferentiated mammalian cells in culture. These cells are currentlyused as sources of natural and engineered proteins and glycoproteins, inscreens for the effects of compounds on cell proliferation and function,and for implantation to supplement or replace cell function. Certaincells are particularly difficult to maintain in long term culture, suchas hepatocytes.

It would be especially useful if hepatocytes could be maintained in longterm culture. For example, in vitro toxicity testing of orallyadministered compounds has been hampered by the fact that the liverconverts many compounds into other chemical forms. These other forms maybe toxic or have other effects. Thus, complete testing of materials incell culture must include the effects of biotransformations carried outby the liver. Using current methodology, it is difficult to grow normalliver cells in vitro beyond two to three cell divisions. The result isthat in vitro testing does not reduce the number of animals neededbecause essentially all of the cells to be used in vitro must come fromdirect isolation. A method of expanding liver cells in vitro would makeit feasible to use in vitro liver cell cultures to carry outbiotransformations by applying the compound of interest directly toliver cells in culture. The supernate from the liver cell cultures couldthen be applied to other types of cells, such as skin, lung, nerve, andbladder, to assess the effect of the metabolized compound of interest.

Studies have been conducted for a number of years to improve theviability, proliferation and differentiated function of eukaryotic cellscultured in vitro. One discovery has been the importance of theextracellular matrix and extracellular matrix molecules in maintainingcell function and allowing cell growth. These effects, and methods ofusing matrix components for cell growth, have been described by, forexample, Jauregui et al., In Vitro Cellular & Developmental Biology 22:13-22 (1986), Kleinman et al., Analytical Biochemistry 166: 1-13 (1987),and Mooney et al., Journal of Cellular Physiology 151: 497-505 (1992).

Growth factors, such as epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), and transforming growth factors (TGFα, TGFβ),exert a broad mitogenic response. Growth factors and their effects havebeen described in "Peptide Growth Factors and Their Receptors I" M. B.Sporn and A. B. Roberts, eds. (Springer-Verlag, New York, 1990). Inrecognition of their importance, most cell and tissue growthcompositions include growth factors, either as an additive or as acomponent of complex growth media. The use of growth factors in thismanner has certain drawbacks. For example, cells have a complex,nonlinear response to the concentration of growth factor in theirenvironment. Extended exposure to high growth factor concentrations maycause cells to lose responsiveness to the factor. For example, EGF, apotent mitogen for a wide variety of cell types and arguably thebest-characterized of the growth factors, when delivered in solubleform, is typically internalized by the cell, and the cell often respondsby down-regulating the number of EGF receptors. This down-regulationcauses cells to lose responsiveness to EGF.

Growth factors have also been used in disappointingly few clinicalproducts, considering the range of effects they produce in vitro.Translation of the mitogenic effects observed for the target cell invitro to tissue growth in vivo is hampered by several issues. Forexample, the growth factors, when placed in a complex cellularenvironment, often end up stimulating the growth of competing cellswhich then overgrow the target cells. Researchers have attempted tosolve this problem by targeting delivery of factors at a specific site,but this approach is not always successful because soluble growthfactors can readily diffuse into the blood stream and away from thetarget site, exerting their effects elsewhere. This diffusion of growthfactors is also a problem because it increases the amount of growthfactor that must be used in order to have the desired local effect.Internalization of growth factors and loss of responsiveness to growthfactors is a particular problem for in vivo applications considering theamount of time cell growth must be stimulated to allow wound healing.

Another attempt to improve the longevity of growth factor effects invivo has been to incorporate growth factors in a slow release material.Such a scheme still requires large amounts of growth factor and does notaddress the problem of competing cell growth due to diffusion of thegrowth factors. The large amount of growth factors needed for these celland tissue growth methods is a particular problem because growth factorsare difficult and expensive to prepare.

It is therefore an object of the invention to provide a cell and tissuegrowth substrate that stimulates long-term target cell growth.

It is another object of the invention to provide a tissue growthscaffold for growth of a target tissue in vivo.

It is a further object of the invention to provide a method of long-termcell and tissue growth in vitro, and to provide a method of growingtarget tissue in vivo.

It is another object of the invention to provide an in vitro tissueanalog for drug and toxicity testing, and a method of drug and toxicitytesting using the tissue analog.

SUMMARY OF THE INVENTION

The methods and compositions described herein concern new cell andtissue growth substrates. Growth effector molecules, including growthfactors and extracellular matrix molecules, are flexibly tethered to asupport medium and the combination is used to stimulate and support celland tissue growth. The most significant feature of these compositions isthat they enhance the biological response to the growth factor. The newcompositions also offer other advantages over the traditional growthmethods, in which growth factors are delivered in soluble form: (1) thegrowth factor is localized to a desired target cell population, and (2)significantly less growth factor is needed to exert a biologic response.In a preferred embodiment, multiple growth factors and/or matrixmaterials are attached to a single core molecule, such as a starpolymer. These compositions can be used as a means of enhancing thetherapeutic use of growth factors in vivo and of creating surfaces whichwill enhance in vitro growth of difficult-to-grow cells such as livercells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of DNA synthesis in cells grown on a non-tetheredsubstrate, with EGF present or absent from the growth medium, plottinglabeling index versus the presence or absence of EGF. The labeling indexis the percentage of cells in a field that have stained nuclei.

FIG. 2 is a graph of DNA synthesis in cells grown with tethered(coupled) or adsorbed EGF, plotting labeling index versus tethered oradsorbed EGF. The labeling index is the percentage of cells in a fieldthat have stained nuclei.

DETAILED DESCRIPTION OF THE INVENTION

Many problems with effective utilization of growth factors may beovercome if, instead of being delivered in soluble form, the growthfactors are immobilized on a solid substrate. This approach isattractive because some forms of insoluble matrix, such as crosslinkedcollagen sponges and bioresorbable polyester fabric, are used for manytypes of tissue regeneration to provide a template for tissue growth.The solid support need not be permanent, and thus the approach may beused for almost any tissue. Immobilization prevents the factor fromdiffusing away from the site and consequently allows a much more highlytargeted form of delivery than other methods. Besides this concentrationeffect, tethering has other powerful advantages, stemming from the waygrowth factors work. For example, when delivered in soluble form, EGF istypically internalized by the cell, and the cell often responds bydown-regulating the number of EGF receptors. However, evidence now showsthat the growth factor does not have to be internalized in order tostimulate cell growth. For example, Reddy et al., Biotechnology Progress10: 377-384 (1994), describes fibroblasts that remain responsive to EGFdespite their expression of internalization-deficient EGF receptors. Asdemonstrated by the following example, by allowing the target cell tobind EGF, but preventing the cell from internalizing the bound EGF, itis possible to circumvent the normal down-regulation of receptors thatoccurs in the presence of high concentrations of EGF. This offers twoadvantages: (1) it is possible to speed the rate of target cell growthin vivo, because cells in contact with the surface bearing the growthfactor do not lose their responsiveness to EGF, and (2) considerablyless growth factor is required, because cells do not internalize anddegrade the growth factor. The method of attachment of the growth factorto the substrate is critical because the receptor must have access tothe factor. Furthermore, for some growth factors, dimerization oraggregation in the membrane is believed to be critical, as described in"Peptide Growth Factors and Their Receptors I" M. B. Sporn and A. B.Roberts, eds. (Springer-Verlag, New York, 1990). Thus, the growth factorwill either have to be immobilized in extremely high concentration orimmobilized on flexible tethers which will allow the ligandreceptorcomplex to aggregate in the cell membrane. Direct immobilization of evenhigh concentrations of growth factor may be ineffective if the receptorsbind randomly.

Tethers

Requirements

As used herein, a tether is a flexible link between an attachmentsubstrate and a growth effector molecule. Flexible tethers for attachinggrowth effector molecules to a substrate must satisfy two importantrequirements: (1) the need for mobility of the ligand-receptor complexwithin the cell membrane in order for the effector molecule to exert aneffect, and (2) biocompatibility of materials used for immobilization.Substantial mobility of a tethered growth factor is critical becauseeven though the cell does not need to internalize the complex formedbetween the receptor and the growth factor, it is believed that severalcomplexes must cluster together on the surface of the cell in order forthe growth factor to stimulate cell growth. In order to allow thisclustering to occur, the growth factors are attached to the solidsurface, for example, via long water-soluble polymer chains, which arereferred to as tethers, allowing movement of the receptor-ligand complexin the cell membrane.

Examples of water-soluble, biocompatible polymers which can serve astethers include polymers such as synthetic polymers like polyethyleneoxide (PEO), polyvinyl alcohol, polyhydroxyethyl methacrylate,polyacrylamide, and natural polymers such as hyaluronic acid,chondroitin sulfate, carboxymethylcellulose, and starch.

Tethers can also be branched to allow attachment of multiple growtheffector molecules in close proximity. Branched tethers can be used, forexample, to increase the density of growth effector molecule on thesubstrate. Such tethers are also useful in bringing multiple ordifferent growth effector molecules into close proximity on the cellsurface. This is useful when using a combination of different growtheffector molecules. Preferred forms of branched tethers are star PEO andcomb PEO.

Star PEO is formed of many PEO "arms" emanating from a common core. StarPEO has been synthesized, for example, by living anionic polymerizationusing divinylbenzene (DVB) cores, as described by Gnanou et al.,Makromol. Chemie 189: 2885-2892 (1988), and Merrill, J. Biomater. Sci.Polymer Edn 5: 1-11 (1993). The resulting molecules have 10 to 200 arms,each with a molecular weight of 3,000 to 12,000. These molecules areabout 97% PEO and 3% DVB by weight. Other core materials and methods maybe used to synthesize star PEO. Comb PEO is formed of many PEO chainsattached to and extending from the backbone of another polymer, such aspolyvinyl alcohol. Star and comb polymers have the useful feature ofgrouping together many chains of PEO in close proximity to each other.

Length

The length of a tether is limited only by the mechanical strength of thetether used and the desired stability of a tethered growth factor. It isexpected that stronger tethers can be made longer than weaker tethers,for example. It is also desirable for tether length and strength to bematched to give a desired half life to the tether, prior to breakage,and thereby adjust the half life of growth factor action. The minimumtether length also depends on the nature of the tether. A more flexibletether will function well even if the tether length is relatively short,while a stiffer tether may need to be longer to allow effective contactbetween a cell and the growth effector molecules.

The backbone length of a tether refers to the number of atoms in acontinuous covalent chain from the attachment point on the substrate tothe attachment point of the growth effector molecule. All of the tethersattached to a given substrate need not have the same backbone length. Infact, using tethers with different backbone lengths on the samesubstrate can make the resulting composition more effective and moreversatile. In the case of branched tethers, there can be multiplebackbone lengths depending on where and how many growth effectormolecules are attached. Preferably, tethers can have any backbone lengthbetween 5 and 50,000 atoms. Within this preferred range, it iscontemplated that backbone length ranges with different lower limits,such as 10, 15, 25, 30, 50, and 100, will have useful characteristics.

Such tethers are not intended to be limited by the manner in which thesubstrate-tether-growth effector molecule composition is assembled. Forexample, if linker molecules are attached to the substrate and thegrowth effector molecule, and then the linkers are joined to form thetethered composition, the entire length of the joined linkers isconsidered the tether. As another example, the attachment substrate may,by its nature, have on its surface protruding molecular chains. If alinker molecule is attached to the substrate via such protruding chainsthen the chain and linker together are considered to be a tether.

Biocompatible polymers and spacer molecules are well known in the artand most are expected to be suitable for forming tethers. The onlyimportant characteristics are biocompatibility and flexibility. That is,the tether should not be made of a substance that is cytotoxic or, inthe case of in vivo uses, which causes significant allergic or otherphysiological reaction when implanted. The tether should also allow thegrowth factor a sufficient range of motion to effectively bind to a cellsurface receptor.

The biodegradability of a tether, the tether-substrate link, or thetether-growth factor link can be used to regulate the length of time agrowth factor stimulates growth. For example, if a given tether degradesduring cell growth at a consistent rate, then a limit can be placed onhow long the growth factors binds to and stimulates cell growth. Onceuntethered, a growth factor can be internalized by the cell or candiffuse away from the target cells. Such planned degradation isespecially useful in the context of implanted compositions, used tostimulate tissue replacement, by limiting the amount of tissue growth.

Attachment Substrates

There are two basic types of substrates onto which growth effectormolecules can be tethered. One class includes biocompatible materialswhich are not biodegradable, such as polystyrenes, polyethylene vinylacetates, polypropylenes, polymethacrylates, polyacrylates,polyethylenes, polyethylene oxides, glass, polysilicates,polycarbonates, polytetrafluoroethylene, fluorocarbons, nylon, siliconrubber, and stainless steel alloys. The other class of materialsincludes biocompatible, biodegradable materials such as polyanhydrides,polyglycolic acid, polyhydroxy acids such as polylactic acid,polyglycolic acid, and polylactic acid-glycolic acid copolymers,polyorthoesters, polyhydroxybutyrate, polyphosphazenes,polypropylfumerate, and biodegradable polyurethanes, proteins such ascollagen and polyamino acids, and polysaccharides such asglycosaminoglycans, alginate, and carageenan, bone powder orhydroxyapatite, and combinations thereof. These biodegradable polymersare preferred for in vivo tissue growth scaffolds. Other degradablepolymers are described by Engleberg and Kohn, Biomaterials 12: 292-304(1991).

Attachment substrates can have any useful form including bottles,dishes, fibers, woven fibers, shaped polymers, particles andmicroparticles. For in vitro cell growth, the growth effector moleculecan be tethered to standard tissue culture polystyrene petri dishes.Woven fibers are useful for stimulating growth of tissue in the form ofa sheet, sponge or membrane.

The biodegradability of a substrate can be used to regulate the lengthof time the growth factor stimulates growth and to allow replacement ofimplanted substrate with new tissue. For this purpose the substrate withtethered growth effector molecules can be considered a scaffold uponwhich new tissue can form. As such, a degradable scaffold is broken downas tissue replacement proceeds. Once released from the substrate, agrowth factor can be internalized or can diffuse away from the targetcells. Such planned degradation is especially useful in the context ofimplanted compositions, used to stimulate tissue replacement, bylimiting the amount of tissue growth and eliminating the need to removethe tissue scaffold. For implantation in the body, preferred degradationtimes are typically less than one year, more typically in the range ofweeks to months.

In some embodiments, attachment of the cells to the substrate isenhanced by coating the substrate with compounds such as extracellularmembrane components, basement membrane components, agar, agarose,gelatin, gum arabic, collagen types I, II, III, IV, and V, fibronectin,laminin, glycosaminoglycans, mixtures thereof, and other materials knownto those skilled in the art of cell culture.

Growth Effector Molecules

Growth effector molecules, as used herein, refer to molecules that bindto cell surface receptors and regulate the growth, replication ordifferentiation of target cells or tissue. Preferred growth effectormolecules are growth factors and extracellular matrix molecules.Examples of growth factors include epidermal growth factor (EGF),platelet-derived growth factor (PDGF), transforming growth factors(TGFα, TGFβ), hepatocyte growth factor, heparin binding factor,insulin-like growth factor I or II, fibroblast growth factor,erythropoietin, nerve growth factor, bone morphogenic proteins, musclemorphogenic proteins, and other factors known to those of skill in theart. Additional growth factors are described in "Peptide Growth Factorsand Their Receptors I" M. B. Sporn and A. B. Roberts, eds.(Springer-Verlag, New York, 1990), for example.

Growth factors can be isolated from tissue using methods know to thoseof skill in the art. For example, growth factors can be isolated fromtissue, produced by recombinant means in bacteria, yeast or mammaliancells. For example, EGF can be isolated from the submaxillary glands ofmice and Genentech produces TGF-β recombinantly. Many growth factors arealso available commercially from vendors, such as Sigma Chemical Co. ofSt. Louis, Mo., Collaborative Research, Genzyme, Boehringer, R&DSystems, and GIBCO, in both natural and recombinant forms.

Examples of extracellular matrix molecules include fibronectin, laminin,collagens, and proteoglycans. Other extracellular matrix molecules aredescribed in Kleinman et al. (1987) or are known to those skilled in theart. Other growth effector molecules useful for tethering includecytokines, such as the interleukins and GM-colony stimulating factor,and hormones, such as insulin. These are also described in theliterature and are commercially available.

The specific function or effect of a growth effector molecule does notlimit its usefulness in the disclosed compositions and methods. This isbecause tethering of a growth effector molecule is used to prevent lossof effect caused by diffusion away from a target cell and/orinternalization of a growth factor.

Only those growth effector molecules that can exert an effect whiletethered are useful in the disclosed compositions. Such an effect,however, need not be the same effect or require the same concentrationas the untethered growth effector molecule. So long as a growth effectormolecule can exert any desired growth effect on a cell while tethered itis considered to be useful for tethering. These useful effects can bedetermined by tethering a selected growth effector molecule andobserving the effect on cell growth using growth assays, such as thosedescribed in the examples below.

Attachment Methods

Standard immobilization chemistries, which are well known in the art,can be used to covalently link the tethers to the growth effectormolecule and the substrate. Tethering growth effector molecules can beaccomplished by attachment, for example, to aminated surfaces,carboxylated surfaces or hydroxylated surfaces using standardimmobilization chemistries. Examples of attachment agents are cyanogenbromide, succinimide, aldehydes, tosyl chloride, avidin-biotin,photocrosslinkable agents, epoxides and maleimides. A preferredattachment agent is glutaraldehyde. These and other attachment agents,as well as methods for their use in attachment, are described in"Protein immobilization: fundamentals and applications" Richard F.Taylor, ed. (M. Dekker, New York, 1991). Growth effector molecules canbe tethered to a substrate by chemically cross-linking a tether moleculeto reactive side groups present within the substrate and to a free aminogroup on the growth effector molecule. For example, synthetic EGF may bechemically cross-linked to a substrate that contains free amino orcarboxyl groups using glutaraldehyde or carbodiimides as cross-linkeragents. In this method, aqueous solutions containing free tethersmolecules are incubated with the substrate in the presence ofglutaraldehyde or carbodiimide. For crosslinking with glutaraldehyde thereactants can be incubated with 2% glutaraldehyde by volume in abuffered solution such as 0.1M sodium cacodylate at pH 7.4. Otherstandard immobilization chemistries are known by those of skill in theart and can be used to join substrates, tethers, and growth effectormolecules.

For the disclosed cell growth compositions, growth effector moleculesmay be tethered either alone or in combinations. For example, bothinsulin and EGF may be tethered to the same substrate. The growtheffector molecules may be combined in any desired proportions. Therelative amounts of different growth effector molecules can becontrolled, for example, by first separately linking the growth effectormolecules to tethers, then mixing the "loaded" tethers in the desiredproportions and attaching them to the substrate. The proportion of eachgrowth effector molecule tethered to the substrate should match theproportion of loaded tethers in the attachment reaction.

Tethering to Aminated Surfaces.

Cell culture surfaces bearing primary amines can be prepared, forexample, by amino-siloxane treatment of glass using reagents which canbe commercially purchased and applied to standard laboratory glasswareor by plasma discharge treatment of polymers in an ammonia environment.Collagen matrices for tissue regeneration have primary amines present inlysine side chains and the terminal amines of each molecule. Twoapproaches are possible. Polymers such as PEO tethers can be activatedon both ends with a leaving group such as tresyl chloride which reactswith primary amines. No blocking is necessary because only the terminalhydroxyl residues of tethers are reactive. This type of reaction can becarried out using standard glassware in a chemical fume hood. Vacuumdrying of the product is required as an intermediate step. A substantialexcess of the activated tether over the number of available amines,dissolved in a saline buffer, is added to the surface to be modified andthe coupling reaction is allowed to proceed. Use of an excess ofactivated PEO in this step minimizes the reaction of both ends of PEOwith available amines and ensures that a substantial fraction ofunreacted activated chain ends are left for reaction with the growthfactor. Unreacted PEO is washed away, and the EGF is then added insaline solution to react with the remaining activated chain ends. Ifmouse EGF is used, only the terminal amino acid is reactive because itcontains no other primary amines. Human EGF contains three possibleimmobilization sites. After the reaction is completed, excess unreactedgrowth factor is removed. This first approach is preferred for attachingEGF to a matrix such as crosslinked collagen, which contains a largenumber of free hydroxyls and which does not allow significantnon-specific adsorption of EGF.

A second approach is to activate the tethers on only one end initiallyby using a substoichiometric amount of activating agent. This will yielda distribution of species which include completely unactivated tether aswell as tether activated at both ends. Unactivated tether can easily bewashed away after the attachment step. The tether is then coupled to thesupport as described above, and the free tether ends are then activatedto allow attachment of EGF. This second approach is preferred forderivatization of cell culture surfaces, which might allow substantialnon-specific adsorption of growth factor, because an intermediate stepin which unreacted amines are blocked with short-chain monomethoxy PEOcan be added before EGF attachment in order to minimize non-specificadsorption of the factor. Cells.

Cells to be cultured using the disclosed compositions can be any cellsthat respond to growth factors or that need growth effector molecule forgrowth. For example, cells can be obtained from established cell linesor separated from isolated tissue. Cells types that can be used with thetethered growth effector molecule compositions include most epithelialand endothelial cell types, for example, parenchymal cells such ashepatocytes, pancreatic islet cells, fibroblasts, chondrocytes,osteoblasts, exocrine cells, cells of intestinal origin, bile ductcells, parathyroid cells, thyroid cells, cells of theadrenal-hypothalamic-pituitary axis, heart muscle cells, kidneyepithelial cells, kidney tubular cells, kidney basement membrane cells,nerve cells, blood vessel cells, cells forming bone and cartilage, andsmooth and skeletal muscle. The cells used can also be recombinant.Methods for gene transfer are well known to those skilled in the art.

In Vitro Cell And Tissue Growth Using Substrates With Tethered GrowthEffector Molecules

Substrates with tethered growth effector molecules can be used toimprove in vitro culture of hard-to-grow cells such as liver cells.Liver cell cultures would be useful for toxicology testing to replacecertain aspects of animal testing of drugs. Liver cells grow very poorlyin vitro using prior art methods, typically undergoing only one or tworounds of DNA synthesis after they are placed in culture. Since atethered growth factor cannot be internalized, tethering will change theway the cells respond to the factor, constantly stimulating them togrow.

Cells can be cultured with tethered growth effector moleculecompositions using any of the numerous well known cell culturetechniques. Standard cell culture techniques are described in Freshney,"Cell Culture, a manual of basic technique" Third Edition (Wiley-Liss,New York, 1994). Other cell culture media and techniques well known tothose skilled in the art can be used with the disclosed compositions.The disclosed compositions are adaptable to known cell culture vessels.For example, growth effector molecules can be immobilized on standardtissue culture polystyrene and glass petri dishes, T-flasks, rollerbottles, stackable chambers, and filter systems such as the MilliporeMILLICELL™ inserts, hollow fiber reactors and microcarriers. Cells canalso be cultured in suspension using the disclosed compositons bytethering growth effector molecules to tiny beads or fibers, on theorder of 10 microns in diameter of length. Such tiny particles, whenadded to culture medium, would attach to cells thereby stimulating theirgrowth and providing attachment signals. The only critical difference inculturing technique is the elimination of growth factor from the cellculture medium when using tethered growth factor compositions. Asdescribed in the examples below, using soluble versus tethered EGF inprimary hepatocyte cultures show an enhanced DNA synthesis rate of thetethered growth factor in comparison to the soluble growth factor. Thiseffect is dependent on the amount of the immobilized factor.

In Vivo Tissue Growth Using Tissue Growth Scaffolds With Tethered GrowthEffector Molecules

In yet another embodiment of the present invention, erodible andnonerodible artificial matrices with tethered growth effector moleculesmay be used either alone or in combination with attached cells toremodel tissue architecture or to repair tissue defects and wounds.

Known methods and compositions for culturing cells and implanting theminto the body can be adapted to use tethered growth effector molecules.For example, U.S. Pat. No. 4,352,883 to Lim, uses cells that areencapsulated within alginate microspheres, then implanted. Suchmicrospheres can be modified with tethered growth effector molecules toimprove their usefulness. Culturing cells on a matrix for use asartificial skin, as described by Yannas and Bell in a series ofpublications, can also be modified by tethering growth effectormolecules to the matrix. U.S. Pat. No. 4,485,097 to Bell, U.S. Pat. No.4,060,081 to Yannas et al., and U.S. Pat. No. 4,458,678 to Yannas et al.describe substrates for use as artificial skin. U.S. Pat. No. 4,520,821to Schmidt describes a similar approach that was used to make linings torepair defects in the urinary tract.

Vacanti et al., Arch. Surg. 123: 545-549 (1988), describes a method ofculturing dissociated cells on biocompatible, biodegradable matrices forsubsequent implantation into the body. Cima and Langer, "TissueEngineering" Chem. Eng. Prog. 89: 46-54 (1993), describe importantconsiderations for the nature and form of implanted matrices useful forinducing tissue replacement. U.S. patent application Ser. No. 08/200,636entitled "Tissue Regeneration Matrices by Solid Free Force Fabrication"filed Feb. 23, 1994 by Cima and Cima, now U.S. Pat. No. 5,518,680, whichis hereby incorporated by reference, describes tissue regenerationmatrices, fabrication techniques, and methods of regenerating tissue. Ingeneral, tissue regeneration devices can be constructed from polymers,ceramics, or from composites of ceramics and polymers. Common materialsuseful for constructing tissue regeneration devices are, for example,extracellular matrix proteins, especially collagens; degradablepolyesters, such as polylactic acid, polyglycolic acid, co-polymers ofpolylactic acid and polyglycolic acid, and polycapralactone;polyhydroxybutyrate; polyanhydrides; polyphosphazenes; bone powder;natural polysaccharides, such as hyaluronic acid, starch, and alginate;hydroxyapatite; polyurethanes; and other degradable polymers describedby Engleberg and Kohn, Biomaterials 12: 292-304 (1991). All of theseknown compositions can be modified by tethering growth effectormolecules to the substrate.

Growth effector molecule tethered compositions for in vivo use can be inthe form of polymeric, attachment molecule-coated sutures, pins, wounddressings, fabric, and space-filling materials. Attachment substratesthat promote ingrowth of dermal fibroblasts and capillaries could alsobe used for dermatological applications and cosmetic surgery, such asrepair of wrinkles and aging skin, burn therapy, or skin reconstructionfollowing disfiguring surgery. Substrates with tethered growth effectormolecules that promote osteoblast migration could be used to fill bonedefects following tumor surgery or for non-healing fractures. Substrateswith tethered growth effector molecules that promote muscle cell growthand migration could be used for replacement of muscle mass, includingcardiac muscle and smooth muscle, following disfiguring surgery and forpatients with muscle degeneration or dysfunction. Tubular substrateswith tethered growth effector molecules that promote growth, migration,and function of epithelial, endothelial and mesenchymal cells can beused for construction of artificial ducts for carrying bile, urine,gases, food, semen, cerebrospinal fluid, lymph, or blood. Sheaths formedof substrates that promote growth of fibroblasts from perichondrium,periosteum, dura mater, and nerve sheaths may be used to recreate thesestructures when they are injured or lost due to surgery or cancer. Inall of these embodiments, either the substrate with tethered growtheffector molecules or substrate plus attached cells may be used forreconstruction in vivo.

Substrates for promoting tissue generation can be formed to have adesired tissue shape. As used herein, a desired tissue shape is theshape that the newly generated tissue is desired to have. For example,certain tissues may need to be sheet-like, tubular, or formed as a lobe.

Deactivation of the growth factor once appropriate tissue regenerationhas occurred can be accomplished by tethering the growth factor to asupport which slowly degrades. Examples of such support materials arepolylactide-co-glycolide and crosslinked hyaluronic acid or collagen.Properly shaped substrate with tethered growth effector molecules can beapplied in clinical problems such as healing of skin or periodontalligament by encouraging continued tissue growth for the life of ashaped, degradable implant.

The disclosed compositions can be administered to animals in variousmodes, including implantation, injection, and infusion. Knownimplantation techniques can be used for delivery of many different celltypes to achieve different tissue structures. The tethered growtheffector molecule compositions may be implanted in many different areasof the body to suit a particular application.

Drug And Toxicity Testing Using Tissue Grown In Vitro On TetheredSubstrates

In another embodiment, cells are cultured on substrates with tetheredgrowth effector molecules and the resulting cell cultures are used toscreen compounds for effects on cell growth, cell proliferation, cellmetabolism, and DNA. For example, the cultured cells can be used toscreen for compounds that alter hepatocyte enzyme systems. The culturedcells can also be used to study metabolism of various compounds and thecarcinogenicity or mutagenicity of compounds both before and aftermetabolism by the cells.

Classically, compounds have been assayed for mutagenic activity usingshort term tests (STT) employing bacterial cell systems or animalstudies. Most animal studies are conducted using the protocol forrodents developed by the National Cancer Institute in the early 1970sand reported by Sontag et al., in U.S. Dep. Health Educ. Welfare Publ.(NIH) Carcinog. Tech. Rep. Serv. 1: 76 (1976). Four STIs that areroutinely used are the Salmonella mutagenesis, SAL, described by Haworthet al., Environ. Mutagen. 5 (suppl. 1): 3 (1983) and Mortelmans et al.Toxicol. Appl. Pharmacol. 75: 137 (1984); chromosome aberrations inChinese hamster ovary cells, ABS; sister chromatid exchanges in Chinesehamster ovary cells, SCE, both described by Galloway et al. Environ.Mutagen. 7: 1 (1985); and mouse lymphoma cell, MOLY, assays,described byMyhr et al., "Evaluation of Short-Term Tests for Carcinogens: Report ofthe International Programme on Chemical Safety's Collaborative Study onin vitro Assays" vol. 5 of Progress in Mutation Research Series, pages55-568, Ashby et al., Editors (Elsevier, Amsterdam, 1985).Unfortunately, the correlation between the rodent assays and the STTs ispoor, and the available STTs do not provide a method for testingcompounds for toxicity or mutagenicity of normal organ-specific cells,nor the effect of metabolism on the compounds by the organ-specificcells, such as hepatocytes.

When testing the effect of potential toxins, control assays using knowntoxins are used for comparison. Examples of known hepatotoxins, such asacetaminophen, carbon tetrachloride, alcohol, and cell-specific virusessuch as hepatitis viruses, can be used to test the suitability of themodel tissue. Standard cell number or cell lysis assays, such as Lactatedehydrogenase release, can be used to measure toxicity. Numerous othertoxicity and mutagenesis assays are known in the art and can bepracticed using cell cultures grown on the tethered growth effectormolecule substrates described herein.

The disclosed compositions can be used to grow liver cells in vitro andmake it feasible to use in vitro liver cell cultures to carry outbiotransformations by applying the compound of interest directly toliver cells in culture. The supernate from the liver cell cultures canthen be applied to other types of cells, such as skin, lung, nerve, andbladder, to assess any derived effect of the compound of interest. Anautomated system which pumps culture medium through a liver cell cultureand then to cultures of these other cell types can be used.

The present invention is further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Enhancement of Cell Growth

Cell Growth And Cell Growth Assessment Methods

A. In Vitro Hepatocyte Culture System.

Rat hepatocytes were prepared according to Cima et al., Biotechnologyand Bioengineering 38: 145-158 (1991). Briefly, rat livers were perfusedwith calcium-free perfusion buffer followed by perfusion buffer withCaCl₂ and collagenase until the livers became soft. Cells were dispersedin William's Medium E supplemented with 10 ng/mL EGF (CollaborativeResearch), 20 mM pyruvate (Gibco), 5 nM dexamethasone (Sigma), 20 mU/mLinsulin (Gibco), 100 U/mL Penicillin/Streptomycin (Gibco). Cells weregrown in culture generally as described by Cima et al. (1991). Briefly,cells were plated in culture medium at a concentration of 3×10⁴ viablecells per square centimeter of culture surface area. Followingattachment, the medium was changed to remove unattached cells and thencells were maintained in medium with daily medium changes. The baseculture medium for growth on tethered substrates and control substrateswas William's Medium E supplemented with 0.55 g/L sodium pyruvate, 0.5pM dexamethasone, 0.8 mg/mL insulin (bovine), 100 U/mLPenicillin/Streptomycin, and 2 mM L-glutamine. In some cases, the mediumwas supplemented with EGF.

B. Quantitative Dot-Blot Assay.

Secretion rates for the proteins albumin, transferrin, fibrinogen, andfibronectin from the hepatocyte cultures were measured with aquantitative dot-blot assay. Media samples from the cultures wereserially diluted and loaded in duplicate onto nitrocellulose paper with0.1 micron pore size using a 96 well minifold apparatus(Schleicher-Schuell). Protein standards were also loaded in duplicate atdecreasing levels from 300 to 10 ng/dot. The blot was then exposed to anappropriate primary antibody for the protein being quantitated. Pabbitanti-rat albumin and anti-rat transferrin were available from Cappel.Rabbit anti-rat fibrinogen was available from Sigma. The non-boundprimary antibody was washed away after one hour, and the blot wasexposed to donkey anti-rabbit IgG labelled with ¹²⁵ I (Amersham) for anadditional hour. The non-bound secondary antibody was washed away, andan autoradiograph of the blot was made. The dots were then punched outand bound ¹²⁵ I measured using a gamma counter to determine the totalamount of bound antibody. A calibration curve was generated by relatingknown amounts of standard protein to total count per minute bound. Thelinear portion of the standard curve was then used to quantitate theamount of protein in the unknown media samples. Secretion rates werenormalized for cell number before the modulating effects of differentattachment molecule densities were compared.

C. One Dimensional SDS-PAGE of Secreted Proteins.

The pattern of protein secretion from cultured hepatocytes wasdetermined by pulse labelling cultures from 46 to 48 hourspost-attachment with ³⁵ S labelled methionine (ICN) in methionine freeWilliam's E media (Gibco), with or without EGF. The media was collectedafter the two hour labelling, and equal amounts of protein were analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Autoradiographs were prepared with XOMAT-XAR5 film.

D. DNA Synthesis Measurement.

DNA synthesis is used as a measure of potential for cellularproliferation. Hepatocytes were pulse labelled for 20 hours beginning at48 hours post-cell attachment with bromedeoxyuridine (BrdU), andsubsequently fixed as outlined above. Cells were processed forimmunocytochemistry using a BrdU kit from Amersham. Briefly, nuclei werepermeabilized with DNAse I during incubation with the primary antibody.Detection of the bound antibody was achieved using peroxidase conjugatedantibody to mouse immunoglobulin, and polymerizing diaminodenzidine(DAB) in the presence of cobalt and nickel, giving black staining atsites of BrdU incorporation. Alternatively, hepatocytes are pulselabelled for 16 hours beginning at 48 hours post-cell attachment with ³H-thymidine, and subsequently fixed in 95% ethanol/5% acetic acidfixative for several hours. The dishes or slides were coated with KodakNTB2 autoradiography emulsion, and allowed to expose for seven days.Autoradiographic grains were developed using Kodak D-19 developer. Thepercentage of cells actively synthesizing DNA was quantitated bychoosing 8 random areas on each dish and counting those cells withlabelled nuclei versus the total number of cells. A minimum of 35 cellswas counted per dish.

Synthesis of growth substrate using polyethylene oxide tether

A. Silyation Reaction.

Glass microscope slides were cleaned by immersion in 1:1 methanol:HCIfor at least 30 minutes. They were rinsed twice in water and immersed in1:1 water:concentrated sulfuric acid for at least 30 minutes. Afteranother rise in water, the slides were placed in boiling water for 15 to30 minutes. In a glove box under a nitrogen atmosphere, the freshlycleaned slides were placed in a solution of freshly mixed acidicmethanol (1.0 mM acetic acid in methanol), 5.0% H₂, and 1% ETDA(N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane) for 15 minutes, andthen rinsed three times in methanol. Following the fmal rinse the slideswere baked on a 120° C. oven for 5 to 10 minutes. The slides were storedin a desiccator at room temperature while awaiting polymer grafting.

B. Activation Of Polymer.

Star polyethylene oxide was dissolved in methylene chloride (10 wt %)and dried over molecular sieve at 4° C. 110 microliters drytriethylamine and 75 microliters tresyl chloride were added to the drypolymer solution for every gram of polymer. After 90 minutes the solventwas evaporated under vacuum and the polymer was redissolved in acidifiedmethanol (0.06M HCl in methanol) and allowed to precipitate at -20° C.To remove unreacted tresyl chloride, the polymer was re-precipitated sixtimes, after which the solvent was evaporated and the dried activatedpolymer stored under nitrogen.

C. PEO Grafting And Reactivation.

Slides were grafted with star polyethylene oxide by placing a droplet of0.1 to 10 wt % tresyl chloride activated polymer in 0.1M phosphatebuffer (pH 7.4) on each slide and allowing the reaction to proceed for12 hours. The slides were rinsed in phosphate buffer and then in water.Slides were dried in graded ethanol solutions of, sequentially, 25%,50%, 75%, and 100% ethanol. Then the slides were rinsed in dry acetoneand finally in dry methylene chloride before reactivation. To tresylactivate the grafted star PEO, slides were immersed for 1 hour in 0.06Mtresyl chloride, 0.07M triethylamine in methylene chloride at roomtemperature under a dry nitrogen atmosphere. For mock activationcontrols, the tresyl chloride was omitted.

D. EGF Coupling And Desorption.

¹²⁵ I-EGF of murine origin was coupled to activated slides in 0.01Mphosphate buffer (pH 7.4) for 12 hours at room temperature. The sameprocedure was followed for control slides. Adsorbed EGF was desorbed bysuccessive washes in 0.01M phosphate buffer (pH 7.4) with 0.1 wt %bovine collagen. The amount of EGF associated with the slides wasdetermined using a gamma counter. The amount of EGF coupled to activatedslides was determined by subtracting the amount adsorbed to the controlslides from that associated with the activated slides.

Growth of cells on tethered substrate in vitro

Freshly isolated rat hepatocytes were seeded on tethered EGF slides andcontrol slides, prepared as described above. The seeded slides wereincubated in William's Medium E supplemented with 0.55 g/L sodiumpyruvate, 0.5 pM dexamethasone, 0.8 mg/mL insulin (bovine), 100 U/mLPenicillin/Streptomycin, and 2 mM L-glutamine. Cells were labelled withbromedeoxyuridine (BrdU) as described above.

Cell seeding and DNA synthesis assays were performed on slides that hadbeen activated with tresyl chloride and coupled with EGF, on mockactivated control slides, both prepared as described above, and oncontrol tissue-culture treated polystyrene dishes (Falcon) either withEGF added to the medium at 10 ng/mL or with EGF omitted.

The results of the DNA synthesis assay for the latter, non-tetheredcontrols is shown in FIG. 1. The presence of EGF in the medium clearlycauses an increase in the number of cells synthesizing DNA. The resultsof the DNA synthesis assay for the tethered EGF surface and the mockactivated control surface that had only adsorbed EGF is shown in FIG. 2.The number of cells synthesizing DNA is clearly higher for the tetheredEGF surface.

Modifications and variations of the compositions and methods of thepresent invention will be obvious to those skilled in the art from theforegoing detailed description. Such modifications and variations areintended to come within the scope of the appended claims.

We claim:
 1. A method for growing eukaryotic cells comprising(a)bringing into contact the cells and a composition comprisingabiocompatible solid substrate, biocompatible branched water solublepolymeric tethers, and growth effector molecules,wherein one end of eachtether is covalently linked to the substrate, each tether is able tocovalently link more than one growth effector molecule, each growtheffector molecule is covalently linked to a distal end of a tether sothat the growth effector molecule cannot be internalized by cellsattached to the substrate, and the growth effector molecules areattached to the substrate in a concentration effective to enhance therate of target cell growth over the rate of target cell growth withsoluble growth effector molecules and growth effector molecules adsorbedto a substrate, without internalization of the molecules; and (b)maintaining the contacting cells and composition under conditions andfor a time sufficient to cause the cells to grow; wherein the step ofbringing into contact comprises administering the composition to apatient in need of cell growth.
 2. The method of claim 1 wherein thecomposition is administered by injection, infusion, or implantation. 3.The method of claim 2 wherein the composition is administered byimplantation of the composition and wherein the substrate is shaped tomatch a desired tissue shape.
 4. The method of claim 3 wherein thesubstrate is biodegradable.